Yeast Two-hybrid Screening For Deubiquitinating Enzymes Interacting With LHBs Protein Of HBV

Hepatitis B virus(HBV)infection is the leading cause of acute and chronic hepatitis,cirrhosis,and hepatocellular carcinoma worldwide.HBV contains four open reading frames of C,S,P and X.The three proteins encoded by S region,namely small proteins(SHBs),medium proteins(MHBs)and large proteins(LHBs),form a viral envelope.Among them,LHBs are closely related to the occurrence and development of HBV-related liver cancer,but the regulation mechanism of LHBs protein levels has not been reported yet.In addition to the transcriptional regulation and protein synthesis,the balance of ubiquitination and deubiquitination is a very important regulatory mechanism of protein level.The aim of this study was to investigate the deubiquitinating enzymes that affect the level of LHBs by protein interactions.Matchmaker Gold Yeast Two-Hybrid System was used to 74 deubiquitinating enzymes interacting with the pGBKT7-LHBs recombinant plasmid.Ten deubiquitinating enzymes interacting with LHBs were obtained,including OTU Domain-Containing Ubiquitin Aldehyde-Binding Protein 1(OTUB1),COP9 Signalosome Subunit 5(COPS5),COP9 Signalosome Subunit 6,COPS6,Ubiquitin Specific Peptidase 26,USP26),Ubiquitin Specific Peptidase 29(USP29),Ubiquitin Specific Peptidase 38(USP38),Ubiquitin Carboxyl-Terminal Hydrolase,BAP1),Deubiquitinating Enzyme EIF3f(EIF3S5),Ataxin-3-Like Protein(ATXN3L),Ubiquitin C-Terminal Hydrolase L5(UCHL5).It was verified that the ten deubiquitinating enzymes did not self-activate in this yeast two-hybrid system,and could also express the corresponding GAL4-AD fusion-expressing protein.Whether the above deubiquitinating enzymes have effects on the LHBs protein level was examined,and the results showed that OTUB1 up-regulated the LHBs protein expression level.GST pull down and mammalian two-hybrid assays were used to verify whether OTUB1 interacted with LHBs.It was found that OTUB1 and LHBs interacted in vitro and in vivo,which lays a foundation for subsequent researches.